Title Morphological Study on the Inhibitory Effect of Germanium Dioxide on Growth and Development of Brown Algae Author(s) Wang, Xiao Yang Citation 北海道大學理學部海藻研究所歐文報告, 9(1), 33-91 Issue Date 1993-03 Doc URL http://hdl.handle.net/2115/48108 Type bulletin (article) File Information 9(1)_33-91.pdf Instructions for use Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP Morphological Study oR the Inhibitory Effect of Germanium Dioxide on Grewth and Development of Brown Algaei) By WANG XIAo YANG2) lntroduction Germanium dioxide (Ge02) has been generally used in unialgal cultures of various seaweeds as an agent for eliminating diatoms. The concentration which would control diatom growth while allowing other algae to grew norrnally was suggested to be l-10 mg Ge02/l (LEwiN 1966). In fact, many studies on the inhibitory effect of Ge02 on various algae and other organisms have been done from very early. Especially, the toxicity ef gerrnanium (Ge) has been recognized at very low concentrations in many diatoms (WERNER 1966, 1967a, LEwm 1966, TATEwAKi and MizuNo 1979), and Ge is generally recognized as a specific inhibitor of diatoms because no inhibitory effects at low doses were found in Chlorophyta (WERNER 1966, 1967b, LEwiN 1966), Rhodophyta (LEwiN 1966, KiRiTA 1970), Cyanophyta (LEwlN 1966), various fungi, bacteria (LEwiN 1966, WERNER 1967b) and protozoa (LEwiN 1966). However, since McLAcHLAN et al. (1971) reported the inhibition by Ge02 at concentrations of 5 mg/l in their culture experiment of four I}etczts species, many species of brown algae have been found to be affected at Iow germanium concentrations (HopKIN and KAIN 1978, TATEwAKI and MIzuNo 1979, MARm{AM and HAGMEIER 1982). In their studies, TATEwAI<I and MizuNo (1979) found that regardless of the constitution of thallus, all the species of brown algae examined showed smaller growth quantity and some damage in the formation of thallus at less than 1-5 mg Ge02/l concentratiens. This response to Ge was found to be significant in the brown algae and net observed in species ef other macroalgae, such as red and green algae, under the same Ge concentrations. The mechanism of the inhibition of Ge on diatoms has been investigated in detail by several workers and there are good descriptions of the effects on frustule morphology and silicate uptake and incorporation. These workers observed that Ge prevents cell division in diatoms generally by altering wall formation (AzAM et al. 1973, Ci{iAppiNo et al. 1977), and specifically, Ge prevents Silicon uptake and also prevents the metabolism of Si already taken up, which in turn seems to affect I)NA, protein or chlorophyll synthesis (DARLEy and VoLcANi 1969, WERNER l966, 1967a). But most Ge toxicity in diatoms was found to be reversible by 1) Thjs manuscript is a part of a thesjs for a doctor's degree, Hokkaido University (1989). 2) Present Address: 1104 S.W. Columbia #308, Portland, OR 972el, U.S.A. 34 X Z VVkeng addition of Si (WERNER 1967b, LEwlN 1966). All these results show that germanic acid is an analogue of silicic acid; it has been thought to act as a competitive inhibitor of Si metabolism in the diatom. This hypothesis has gained wide acceptance to become a matter of general knowledge. As to the germanium toxicity to brown algae, however, there are only a few explanations and speculations so far (TATEwAI<I and MIzuNo 1979, MARKHAM and HAGMEIER 1982). MARKHAM and HAGMEiER (1982) explained the brittleness due to germanium inhibition of the Laminare'a plants as a result of the competitive action between Si and Ge elements, because PARKER (1969) had reported the presence of Si in brown and green algae and suggested that silica is localized in the cell wall in brown algae. However, IV{cLAcl{LAN et al. (1971) and Mc- LAc}iLAN (1977) have found that the omission of silicon from culture medium had no effect on growth and development of embryos of Fzaczas edenintzas, and excess silicon was not effective in suppressing the effects of germanium toxicity in the species. Similar}y, TATEwAm and MIzuNo (1979) performed an experiment with several brown algae including JF}etczts evanes- cen$ Stytosiphon lomentaria and Desmarestia viridis, to see whether the brown algae show any dependence on Si and whether additional Si will reduce the inhibition of Ge. Their results did not show any effect of Si on the brown algae. Therefore, it can be thought that the brown algae do not require Si as an essential nutrient, and it is possible that another mechanism of Ge inhibition exists rather than competing with Si as an analogue in the brown algae. So the questions are: Why are the brown algae are more sensitive to Ge02 than other macroalgae, how do the brown algae respond to germanium treatment and then how are they affected. In living organisms, all the physiological and biochemical processes are eventually reflected in their morphological features. In this way, the inhibitory processes of Ge on brown algae can be expected to appear also morphologically. Therefore, investigating the changes in morphology after treating with Ge will be helpful in deducing the possible mechanism of Ge inhibition. In order to make the mechanisrn of germanium inhibition on brown algae clear, an examination focused on the morphology of plants and cells has been carried out in the present study. Acknowledgments I wish to express my thanks to Pro£Masakazu TATEwAKI, the Institute of Algological Research, Faculty of Science, Hokkaido University, whose encouragement, guidance and provision of facilities have been the most valuable and helpful throughout this study. I am very thankful to Dr.Taizo MoToMuRA, for his teaching and help in the research with electron microscope. Also the critical comments and kind suggestions frorn him and Dr.Isamu WAKANA, were very useful and greatly appreciated. Finally, I would like to say thanks to the staffs of the Institute of Algological Research, Faculty of Science, Hokkaido University, for their assistance in a variety of ways. kthibitory llffec.t of Germanium Dioxide on Brown itligae 35 Chapter 1. IRhibition of Ge02 in brown algae and their morphology under the light microscope lntroduction A significant growth inhibition due to small amounts of germanium has been observed in many diatoms (LEwlN 1966, WERNER 1966, 1967a, TATEwAKi and MizuNo 1979) and brown algae (McLAc}ILAN et al. 1971, McLAc}iLAN 1977, HopKiN and KAiN 1978, TATEwAi<i and MIzuNo1979,MARI<}IAMandHAGMEIER1982). Boththegroupsofalgaeshowhighersensitiv- ity to germanium compounds. However, differing from the diatoms, in which germanium function has been clarified as a competing analogue with nutrient silicon (Si), the brown algae show no dependence on Si and additional Si does not work in reducing the inhibitory effect of Ge. So, it is difficult to assume that germanium inhibits the brown algae in the same way as in the diatoms. In the brown algae examined so far, the damage to the thallus due to Ge treatment has been observed together with the inhibition of the growth rate (TATEwAKi and MizuNo 1979, MARKHAM and HAGMEIER 1982). Frorn those reports it can be seen that the inorphological change in thallus varies greatly according to the species, which is probably because of the fact that there is a large variation of the system of thallus organization and the way of growth even in the same group of brown algae. It is reasonable to think that relatively earlier affected place is most sensitive to Ge toxicity and this position in the thallus depends on the species. This possible difference in Ge sensitivity location might cause the different appear- ance of thallus when altered by germanium. However, all the brown algae examined have in common higher sensitivity to Ge compared with other macroalgae, no matter how different they appear (TA'rEwAI<I and MizuNo 1979). This generality in brown algae is suggestive of some way of reacting applicable to all the species in the group. The morphological change is thought likely to provide some information about the inhibitory mechanism of Ge02. To know the ways in which various brown algae respond to germanium, and further find the general character in their morphological reaction to germa- nium, observations were done on several brown algae under the light microscope. Materials and Metheds Four species of the brown algae, Fhrczts distichus subsp. evanescens (C. AG.) PowELL, felvetia tvrilg72tii OKAMuRA, Analipus 7'mponicus (HARv.) WyNNE and Stytosiphon lomentaria (LyNGB.) LINK, appears from the upper tide zone to the lower tide zone at Charatsunai, Muroran, Hokkaido. Fertile plants are coininonly found from February to June in Analipus itiponicus and Strytosiphon lomentaria, from April-May to July in Ihrcus distichzts and from September to December in 1lelvetia wrightii. The strains of S)bhacelaria sp. and Pilayella litto7zzlis (L.) KJELLMAN were from the culture collection (see Table l). Mature plants of Iihrczts distichus and Rglvetia w7'ightii were rinsed with autoclaved 36 X. M Pla7rg Table 1. Species used for the experiment Date of Species PIace of collection collection Phaeophyta Eescus distichus subsp. Charatsunai, 1986-1988. evanescens Muroran, Hokkaido Ptelvetia zvrightii Charatsunai, 1986-1988. Muroran, Hokkaido Analipzts 7'oponicus Charatsunai, 1986. 12.- Muroran, Hol<kaido 1988. 7. Sqytosiphon lomenlan'a Charatsunai, 1987. 2.- Muroran, Hokkaido 1988. 7. SPhaceimia sp. Culture collection of Texas Univ. adyella litto7'alis Culture collection at the Institute of Algological research, Hokkaido Univ. seawater and stored at 180C in constant illumination of 3000-400e lx (12-14 W/m2) overnight. After about 4 hr incubation at 40C in a dark condition, eggs and sperms were released from the receptacles by immersion of the plants in cold seawater. Around the released eggs, many swimming sperms could be observed under the light rnicroscope.